Methods of the kind under discussion here have been known in practical use for some time in a variety of embodiments. Of the known methods, only fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), or photoactivation will be mentioned by way of example. It is characteristic of these methods that a definable region of interest (ROI) is illuminated in a particular fashion. In FRAP, FLIP, or photoactivation experiments, this so-called manipulation illumination is characterized, for example, by a particularly high brightness. In the corresponding inverse experiments (inverse FRAP, inverse FLIP, inverse photoactivation), on the other hand, a particularly dark manipulation illumination is selected. Also known are methods in which the illumination during normal imaging and the manipulation illumination are distinguished from one another by their respective spectral compositions.
The purpose of the manipulation illumination is to set in motion certain processes in the specimen being investigated; this can be, for example, bleaching or photoactivation of a fluorophore. Also conceivable is a reorganization within a fluorophore. These processes can result in changes in the spectral properties, or other detectable changes.
By way of a locally differing selected manipulation illumination, local properties can be imparted to the specimen so that, for example, after manipulation illumination certain parts of cells have fluorophores that are visible particularly well or particularly poorly. The local properties imparted to the specimen can then be made visible in a confocal microscope.
A redistribution of the fluorophores takes place as a result of transport processes in the interior of the specimen, for example inside cells. In many cases the transport processes ultimately result in a more or less homogeneous distribution of the fluorophores. The behavior over time of these processes can be made visible with the aid of microscopic images, allowing conclusions as to the transport processes in the specimen.
Conventional experiments of the kind described above are generally carried out in such a way that the specimen is first scanned, once or repeatedly, with a laser light beam for the manipulation illumination. In a subsequent step, the specimen is then scanned for the actual image acquisition. The result obtained therefrom is a series of images at different time intervals. At a typical image acquisition rate, which is on the order of 1 to 100 frames per second, the time interval between two successive images is 10 ms to 1 second. It is extremely problematic in this context that diffusion processes in biological specimens generally proceed much more quickly. A fluorophore typically moves in a few microseconds out of the focus of a confocal microscope. Such snapshots consequently allow only extremely poor investigation of local properties of transport processes, for example local flow directions, barriers, etc., since a local excitation effected by the manipulation illumination propagates too greatly between two successive images. For image production, for example for color depiction of regions of the specimen having different transport properties, the known methods are, for the reasons described, quite entirely unsuitable.
The so-called “volume effect” moreover causes additional problems for specimen investigation. The volume effect becomes apparent by the fact that transport processes also take place physically out of and into the image plane of a confocal microscope. Because the regions of the specimen located outside (i.e. above and below) the image plane are not accessible to observation, it is extremely difficult to interpret measured diffusion constants or other local conditions. For example, a local transport barrier in the image plane is not visible if the transport process overcomes that barrier by bypassing it through the volume located above or below it. Signal quality is also negatively affected by the volume effect, since manipulated fluorophores migrate relatively quickly into the volume above and below the image plane that is inaccessible to observation.
To circumvent the problems associated with the volume effect, measurements for the investigation of transport processes in specimens are often performed with the confocal microscope pinhole open. This degrades the resolution in the laser beam direction, and a projected image of the specimen, instead of a defined section of the specimen, is acquired. This allows better interpretation of the data that are obtained, but at the same time the essential advantages of a confocal microscope—such as high resolution, flare suppression, etc.—must be sacrificed.